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R. Gao et al. / Drug Metabolism and Pharmacokinetics xxx (2015) 1e8
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subjected to glucuronidation reactions were metabolized by UGTs
[10]. However, the metabolic characteristics of UA and which UGT
enzymes responsible for UA remain unclear. Identifying the UGT
enzymes responsible for UAwould help us to characterize the role of
these particular enzymes in potential clinical herb and drug in-
teractions and estimate the impact of genetic polymorphisms of
interesting enzymes on drug disposition.
The purpose of this study is to characterize the glucuronidation
kinetics of UA in HLMs and HIMs and identify the main UGT en-
zymes involved using a battery of recombinant human UGTs. In-
hibition of UGT-glucuronidation activity and correlation in HLMs
were also determined using known high-affinity UGT substrates or
inhibitors to facilitate identification of the UGT enzymes involved in
UA glucuronidation.
100e700, with full and product ion scans, and selected ion moni-
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toring. The operating parameters of the MS were as follows: gas
ꢀ
flow, 8 L/min; gas temperature, 350 C; capillary voltage, ꢂ4000 V;
nebulizer pressure, 40 psi; and fragmentor voltage, 135 V.
2.3. Quantitative analysis of glucuronidation assay of UA
The glucuronidation of UA by HLMs or HIMs was performed as
described above. The quantitative analysis of UA glucuronide was
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achieved by injecting 5
ml of the dried residue redissolved in mobile
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phase into an LCMS-2010 EV system consisting of a Shimadzu LC-
20AB (Shimadzu Corporation, Kyoto, and Japan) system equipped
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with a Luna C18 column (50 mm ꢁ 2.0 mm, 5
m
m). Mobile phase of
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ammonium formate (5 mM, pH 3.41: acetonitrile, 20:80, V/V) was
run at a flow rate of 0.2 ml/min. SIM was performed in the negative
ion mode. The detector voltage was put at ꢂ1.85 kV. The block
heater and the curved desolvation line temperature were put at
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2. Materials and methods
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2.1. Chemicals and reagents
200 C and 250 C, respectively. Other MS detection conditions
were set as follows: interface voltage, 40 V; voltage, 4 kV, drying gas
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Ursolic acid (99.8%) was supplied from Engineering Develop-
ment Center of Yichun College, Jiangxi, and China. UDPGA, heco-
(N
) pressure,60 Pa; and nebulizing gas (N
) flow, tuned to be 1.5 L/
2
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ꢂ
2
1
min. SIM was performed by monitoring the [MeH] m/z of 455.35
for UA and 526.25 for gliquidone (IS).
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genin,
b
-glucuronidase (Helix pomatia), and alamethicin
[SigmaeAldrich Corporation, Saint Louis, Missouri, and United
States of America (USA)]. Pooled HLMs, HIMs and individual HLMs
were provided from Research Institute for Liver Diseases (Shanghai
Corporation Limited, Shanghai, and China). Recombinant human
UGT Supersomes™ (UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10,
2B7, 2B4, 2B15, and 2B17) expressed in baculovirus-infected insect
cells [BD Biosciences, Bedford, Massachusetts (MA), and USA].
As standards of the UA glucuronides were not commercially
available, the amount of each UA glucuronide formed in vitro was
calculated from the removal of parent compound UA. Incubations
with inactivated HLMs or HIMs served as control group. The cali-
bration curve of UA in HLMs or HIMs incubation system were
constructed over 0.5e64
mM and 0.125e16
mM, respectively. There
was a good linearity for the determination of UA in HLMs incuba-
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Magnesium chloride (MgCl
2
) (Tianjin Damao Chemical Reagent
tion system from 0.5 to 64
system from 0.125 to 16
m
M (r ¼ 0.998) and in HIMs incubation
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Factory, Tianjin, and China). Gliquidone, telmisartan, chenodeox-
ycholic acid, and trifluoperazine (TFP) hydrochloride (National
Institute for the Control of Pharmaceutical and Biological Products,
Beijing, and China). Milli-Q water (Millipore Corporation, Billerica,
MA, and USA) was used in all steps, and all other chemicals were of
high-performance liquid chromatography (HPLC) grade or the
Analytical grade which was commercially available.
m
M (r ¼ 0.994).
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For the confirmation of the site of glucuronidation of UA, the
dried residue from above incubation system in HLMs was hydro-
ꢀ
lyzed in 200
m
l of 0.01 mol/L sodium carbonate solution at 45 C for
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2 h. The peak area of UA glucuronidation production after hydrolysis
was detected and compared that without alkaline pretreatment.
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2.4. Hydrolysis with b-glucuronidase
2.2. Qualitative analysis of UA glucuronide by LC-MS/MS
UA was treated with HLMs as described above, and the super-
natant was collected, dried and redissolved in 200 l of 50 mM
sodium acetate buffer (pH 5.27). Each sample was incubated in the
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UA (8
mixture containing 0.5 mg/ml of microsomal protein (from HLMs
or HIMs), alamethicin (50 g/mg protein), UDPGA (3 mM), MgCl
mM) was incubated in a final volume of 200
ml incubation
m
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ꢀ
m
2
absence or presence of 4000 units of b-glucuronidase at 37 C for
(8 mM), Tris-hydrochloric acid (50 mM, pH 7.4). Microsomes were
preincubated with alamethicin for 20 min on ice before incubation.
4 h. Each incubation reaction was terminated as described above,
and the supernatant was injected for LC-MS analysis.
ꢀ
After preincubation at 37 C for 5 min, the reaction was initiated by
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the addition of UDPGA at 37 C for 30 min, the reaction was
terminated with 1 ml of cold ethyl acetate (containing 8 M gli-
2.5. Glucuronidation by recombinant UGTs
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m
quidone used as internal standard for the subsequent quantitative
determination of UA). Then the incubation mixtures were centri-
Twelve commercially available recombinant human UGTs
(UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9,
UGT1A10, UGT2B4, UGT2B7, UGT2B15, and UGT2B17) were used to
screen the glucuronidation of UA at three concentrations (4, 8, and
16 mM). Incubation conditions and analyses were similar to those
HLMs except that the protein concentration was 0.2 mg/ml.
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fuged at 20,000ꢁ g and 4 C for 10 min, the supernatant was
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collected and dried using concentrated drying apparatus at 55 C
for 25 min. The dried residue was redissolved in 200
ml of mobile
phase and 5 l aliquots were analyzed by high performance liquid
m
chromatography-mass spectrometry/mass spectrometry (HPLC-
MS/MS) for metabolite profiling. Incubations without UDPGA and
substrate served as negative controls.
2.6. Kinetic analysis
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LC-MS/MS was performed using Agilent 6430A LC-MS/MS sys-
tem coupled with an Agilent 1200 series HPLC system. Chromato-
graphic separation was achieved on a Luna C18 (50 mm ꢁ 2.0 mm,
UA concentration series from 1 to 64
HLMs, recombinant UGT1A3 and UGT1A4, and UA at different
concentrations from 0.5 to 40 M were treated with pooled HIMs.
mM were performed in
m
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1
5
mm) column with the mobile phase of ammonium formate (5 mM,
Kinetic parameters were evaluated from the suitable curves using
Graphpad Prism (Graphpad Software Inc., California (CA), and USA)
followed by nonlinear regression analysis. The following equation
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pH 3.41) and acetonitrile (20:80, V/V) at a flow rate of 0.2 ml/min.
The pH of ammonium formate (5 mM) was adjusted by formic acid.
Electrospray ionization (ESI) was performed to identify UA glucu-
ronide operating in both negative and positive ion mode from m/z
was used for assuming
a
MichaeliseMenten equation:
V ¼ Vmax ꢁ [S]/([K þ [S]). Where V is the rate of reaction, Vmax is
m
Please cite this article in press as: Gao R, et al., Identification and characterization of human UDP-glucuronosyltransferases responsible for the
Gao, Rui
